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Week 5 - Moving On

I have surprising news to share this week—I made more calibration curves! The curve I made this week was for Thalassiosira pseudonana. It should have gone smoothly since I’ve made a bunch of these by now, but the data looked a little funky. The spectra didn’t have big chlorophyll peaks and the isobestic point was off. Ideally when working with SNARF all the spectra of the different pH should cross at one point which is the isobestic point. The spectra I got all converged around the same point but it wasn’t as close as prior data that I’ve collected. Naturally that got me worried about the accuracy of my result for the curve but because we use ratiometric detection the curve worked out just fine. It’s amazing how taking the ratio between two peaks can cancel out so many errors. This experience has truly taught me to love ratios. My next curve for a freshwater phytoplankton, Chlorella, worked out great.

After making the two calibration curves, I began my transition to move away from the spectrofluorometer. Rachel trained me on the flow cytometer which is another machine that can be used to measure intracellular pH with SNARF. It’s a very fancy machine which does scare me a bit if I stop to think about how expensive it is. I’m still getting comfortable working with equipment that costs more than my life savings all the time. I tend to be a clumsy person, so the threat of me breaking something is very real. Despite that, I do like the flow cytometer because of the way it works. It can count thousands of individual particles in minute which I think is pretty amazing. I am excited to collect more data from it in the coming weeks.

On Friday I worked with Rachel to get the pHstat running. We tried to get it to work the first week I was here but accidently short circuited a board. We ordered a new one but that, of course, got lost when shipped, so it only just got to the lab on Wednesday. It took a bit of rewiring but by the end of the day we got everything to work. I'm hoping everything stays working so we can run some experiments after the weekend.

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